In vitro acaricidal activity of Melia azedarach ripe fruit extract against Hyalomma excavatum (Acari: Ixodidae)

Document Type : Research Article


1 Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

2 Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

3 Laboratory of Hormozgan Veterinary Head Office, Bandar Abbas, Iran.

4 Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.


The current study aimed to evaluate the effect of dichloromethane extract of Melia azedarach ripe fruit on larvae and adult females of Hyalomma excavatum at concentrations of 0.25, 0.5, 1, 2, and 4%, using the larval immersion test (LIT) and adult immersion test (AIT). The results showed that in LIT, the percentage mortality of larvae was significantly higher at concentrations 1, 2, and 4% than the control group after 24 h. While the mortality rates varied from 8.66% to 72.66% after 24 h post-treatment, complete mortality of the examined larvae was achieved at concentration of 4% after 48 h post-exposure whereas, it was13.33% in the negative control group. In AIT, the percentage inhibition of oviposition in the treatment groups was significantly greater than the control group (P < 0.01). The maximum inhibition of oviposition was 17.72%, which was achieved at a concentration of 4% and it was 0% in the control group. The difference between reproductive index in treatment and control groups was not statistically significant (P > 0.01). This study showed that the ripe fruit extract of M. azedarach was toxic to H. excavatum under laboratory conditions.


Main Subjects


DCM: Dichloromethane

LIT: Larval immersion test

AIT: Adult immersion test

AZA: Azadirachtin

IO: Inhibition of oviposition

RI: Reproductive index


Hard ticks (Arachnida: Acarina: Ixodidae) are obligate blood-feeding ectoparasites that affect both human and animal health via sucking blood and transmission of some pathogenic agents such as Babesia spp., Theileria spp., Anaplasma spp., and Nairovirus [ 1 , 2 ]. Anxiety, irritation, stress, skin damage, weight loss, tick paralysis, decrease in milk production, loss of production, and anemia are direct adverse consequences of infestation with the hard ticks [ 2 ].

Nowadays, tick control relies on using synthetic pesticides. Although these compounds are more available and possess fast-killing effects, intensive and repeated use of them to control tick infestations has resulted in developing resistance to an array of acaricides. Rhipicephalus (Boophilus) microplus resistance to permethrin was recorded in the USA and Mexico [ 3 ]. Resistance to cypermethrin and deltamethrin in Hyalomma anatolicum has been reported from India [ 1 , 4 ]. Tick resistance to conventional pesticides and increased demand for organic products has accelerated the research on plant-based acaricides [ 5 ].

Melia azedarach, belonging to the Meliaceae family, is a well-known source of various bioactive components with insecticidal properties. This deciduous tree species native to Indomalaya and Australasia is now cultivated in most subtropical and tropical regions of the world [ 6 , 7 ]. Acaricidal, insecticidal and larvicidal efficacy of M. azedarach extract against agriculture pests, mosquitoes, important veterinary ticks, and mites have already been reported [ 8 - 11 ]. M. azedarach has been reported to have a complex mixture of compounds including saponins, terpenoids, flavonoids, tannins, alkaloids, and limonoids [ 6 ]. Limonoids particularly azadirachtin (AZA) constitute the biologically active components of M. azedarach fruits. However, other limonoids such as nimbin, nimbolinin, and salannin have also been reported from the M. azedarach fruits [ 6 , 12 , 13 ] AZA is the most important limonoid and biopesticide of this plant which its toxicity and adverse effects on feeding, growth, fecundity, and oviposition of arthropods, especially for phytophagous insects have been proven [ 14 - 16 ]. This compound is found in different parts of the M. azedarach tree and the highest level of AZA is generally found in seeds [ 17 ]. Although several methods have been reported for the identification and quantitative determination of azadirachtin, most studies have used high-performance liquid chromatography (HPLC) for this purpose worldwide [ 17 ]. The AZA content in various parts of trees is influenced by several factors such as genetic, climatic conditions, harvesting time, geographical area, and time of collection/storage of plant materials [ 17 ].

Adult Hyalomma excavatum ticks (known as large Anatolian Hyalomma) infesting cattle, sheep, horses, goats, camels, and donkeys are found almost all over Iran except the Caspian Sea area [ 18 - 21 ]. This Hyalomma species serves as a vector for some pathogens particularly Theilera annulata and Razmi et al. (2003) reported a high rate of T. annulata infection in examined H. excavatum collected from cattle in Mashhad area, Iran [ 21 , 22 ].

Due to the high prevalence of H. excavatum in most parts of Iran and the necessity of searching for less hazardous and eco-friendly alternatives for synthetic acaricides, the present study aimed to evaluate acaricidal effects of M. azedarach fruit extract on H. excavatum under laboratory conditions.


In larval immersion test (LIT), the percentage mortality of larvae was significantly higher at concentrations 1, 2, and 4% than the control group after 24 h. The mortality rates varied from 8.66% to 72.66%, 24 h post-treatment, and 100% mortality of examined larvae was achieved at concentrations 4%; 48 h post-exposure. The extract killed 100% of the tick larvae at all concentrations after 72 h, while the mortality rate was 17.30% in the control group (Figure 1, Table 1).

Figure 1. Linear regression curve of percentage mortality of H. excavatum larvae in Probit unit versus logarithm concentration of M. azedarach ripe fruit extract.

Time (h) Control group (water + tween) Case group (M. azedarach extract %)
0.25 0.5 1 2 4
24 3.33±5.77 Aa 8.66 ± 3.21 Aa 15.33 ± 4.50 ABa 29.00 ± 9.64 Ba 58.33 ± 17.55 Ca 72.66±16.16 Ca
48 13.33±7.63 Aa 53.66 ± 9.81Bb 59.33 ± 7.37 Bb 82.00 ± 23.06 Db 89.66 ± 17.89 Db 100.00±0.00 Db
72 17.30±8.32 Aa 100.00±0.00 Bc 100.00±0.00 Bc 100.00±0.00 Bb 100.00±0.00 Bb 100.00±0.00 Bb
Different capital letters within lines and small letters within columns are significantly different (P<0.05).
Table 1.Means ± SD of mortality rates of Hyalomma excavatum larvae in treatment and control groups exposed to different concentrations of M. azedarach ripe fruit extract 24, 48, and 72 h post-exposure.

The LC50 and 99 (lethal concentrations of 50 and 99%) values of this extract against examined tick larvae were calculated at 24 and 48 h post-exposure and is presented in Table 2.

Time (h) Slope (95% CL) R2 LC50 (%) (95% CL) LC99 (%) (95% CL)
24 1.77 ± 0.17 0.97 1.86 (1.77-1.96) 38.40 (36.48-40.32)
48 2.33 ± 0.56 0.85 0.33 (0.31-0.35) 3.32 (3.15-3.48)
CL: Confidence Limit
Table 2.The LC50 and LC99 values of M. azedarach ripe fruit extracts against H. excavatum Larvae.

Adult immersion test

In adult immersion test (AIT), the percentage inhibition of oviposition in treatment groups was significantly greater than the control group (p < 0.01). The maximum inhibition of oviposition was 17.72%, which was achieved at a concentration of 4% and this parameter was concentration-dependent. The difference between reproductive indexes in the treatment and control groups was not statistically significant (p > 0.01). The detailed data about the inhibition of oviposition and reproductive index is presented in Table 3.

Tick reproduction Control group (water + tween) Case group (M. azedarach extract %)
0.25 0.5 1 2 4
Inhibition of oviposition (%) 0.00 ± 0.00 4.14 ± 2.19 6.66 ± 2.89 8.74 ± 3.97a 14.26 ± 6.07a 17.72 ± 6.15a
Reproductive index 0.63 ± 0.06 0.60 ± 0.05 0.59 ± 0.03 0.57 ± 0.03 0.54 ± 0.06 0.52 ± 0.08
a Significant differences compared with the control group (P <0.05).
Table3.Means ± SD of mortality rates of Hyalomma excavatum larvae in treatment and control groups exposed to different concentrations of M. azedarach ripe fruit extract 24, 48, and 72 h post-exposure.


The LIT results demonstrated a different level of larval mortality. Besides, the percentage of mortality of exposed larvae was concentration and time-dependent. These findings confirm several similar studies that investigated the effectiveness of M. azedarach crude extract against ticks, mites, and mosquitoes [ 8 , 10 , 11 ]. Borges et al. (2003) showed that the hexane extract of M. azedarach ripe fruit was effective against Rhipicephalus (Boophilus) microplus larvae in a concentration and time-dependent manner [ 8 ]. The acaricidal effects of this extract against Demanyssus gallinae and Tetranychus urticae have been reported [ 10 , 23 ]. Furthermore, Selvaraj and Mosses (2011) observed that leaf and seed extract of this tree produced significant larval mortality in all larval stages of Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti [ 11 ]. Azadirachtin is recognized as the main active ingredient and the most important component of Azadirachta indica and M. azedarach. Feed deterrency, growth reduction, increase in mortality, abnormal/delayed molts and down-regulation of insects’ reproductive organs have been observed after applying AZA [ 14 ]. The other known bioactive components of M. azedarach include meliartenin, meliacaprin, meliacin, meliantrol, melianol, salannin, nimbin, and pinoresinol bis-epi-pinoresinol with anti-feeding/growth activities, repellency, inhibition of oviposition, and embryogenesis properties [ 24 , 25 ]. In the present study, a high mortality rate in examined tick larvae was observed after a short time post-exposure. This fast-killing effect may be related to inhibition of cell division and protein synthesis in cells of ectoparasites [ 14 ].

In this study, egg production in exposed ticks was significantly lower than that in the control group, and this extract at the concentration of 4% produced 17.72% inhibition of egg production. In a similar study, the ripe fruit extract of M. azedarach at the concentration of 0.25% caused complete egg production inhibition in Boophilus microplus [ 8 ]. Variation in efficacy with different concentrations may be associated with genetic characteristics of the plant, edaphoclimatic conditions, and even duration of storage affecting the chemical composition of a plant extract [ 26 , 27 ]. Reproductive disruption in examined ticks can be attributed to azadirachtin that affects reproductive tissues at molecular/cellular levels and disrupts endocrine processes by altering ecdysteroids and juvenile hormone titers [ 14 ]. Sousa et al. (2013) observed a reduction in ovary weight, morphological changes in oocysts, vacuolization, chorion deformity, and disorganization of the yolk granules of engorged females Rhipicephalus (Boophilus) microplus treated with M. azedarach hexanoic extract [ 28 ]. To fully understand the various acaricidal effects of M. azedarach, further studies are required to identify all its bioactive acaricide components with their special effects at cellular and molecular levels.

Presently, there is scant published data on LC50 values for Hyalomma spp. exposed to M. azedarach extract. The current study recorded an LC50 value of 1.86% for H. excavatum treated with dichloromethane extract of M. azedarach 24h post-exposure. LC50 values of 0.26 and 4.17% were reported for Hyalomma dromedarii nymphal stage exposed to petroleum ether and ethyl alcohol extract of M. azedarach. Also, these extracts showed a significant effect on H. dromedarii eggs with LC50 values of 3.14 and 1.77%, respectively [ 29 ]. LC50 value of 1.78% was recorded for Dermanyssus gallinae tereated with Hexan extract of M. azedarach [ 10 ]. These variations in LC50 values can be attributed to the type of solvent used for plant extraction, susceptibility of exposed ectoparasite species, and its developmental stage.

Besides the laboratory studies, Borges et al. (2005) and Sousa et al. (2011) evaluated the efficacy of hexane extract of ripe fruits of M. azedarach against all developmental stages of R. microplus [ 30 , 31 ]. Standardized laboratory and farm tests need to be developed to add along with this incremental process for finding natural pesticides.

In conclusion, the findings of this study showed that M. azedarach ripe fruit extract was effective against larvae and engorged females of H. excavatum ticks, and more studies are required to investigate its efficacy in field trials.

Collection of ticks

Adult males and females of H. excavatum were taken from an active tick colony rearing in the Faculty of Veterinary Medicine’s parasitology laboratory, Ferdowsi University of Mashhad, Iran. The identification of ticks was made using a stereomicroscope based on morphological criteria [ 21 ].

The developmental stages of ticks

Unfed mixed sex adult ticks were experimentally fed on healthy pathogen-free rabbits (male) at room temperature. 180 adult engorged female ticks were used for adult immersion test (AIT), but a group was placed into tubes individually and kept at 28 ºC, 80% RH to oviposit. Eggs were harvested and divided into groups of 300 eggs and transferred into perforated tubes. Newly hatched larvae were maintained at 28 ºC, 80% RH in an incubator and used for larval immersion test [ 32 ].

Preparation of the crude extract

The collected M. azedarach ripe fruits from the campus of the Ferdowsi University of Mashhad, Mashhad, Iran, were dried in the shade at room temperature and powdered using a grinding machine. The powder was extracted with dichloromethane (DCM) in the Soxhlet extraction apparatus and the solvent was removed by a rotatory evaporator. The residue was serially diluted with distilled water to obtain desired concentrations of 0.25, 0.5, 1, 2, and 4% and Tween 20 was used as emulsifier to ensure complete solubility of the materials in water [ 8 , 10 ].

Larval immersion test

Larval immersion test (LIT) was performed based on the methodology described by Singh et al. (2017) [ 33 ]. Approximately 300 14-21 day old larvae were immersed in 0.5 ml of each desired concentration for 10 min. Then, the larvae were transferred on a filter paper to dry and 100 larvae were taken and placed in a folded filter paper packet (7.0 cm by 7.0 cm) using an aspirator. The pockets were sealed with adhesive tape and incubated at 28 °C and 80% RH. The packets were opened after 24, 48, and 72 h for counting live and dead larvae [ 33 ]. The mortality rate was corrected using the Abbott formula if mortality in the control group was between 0 and 5%:

Corrected mortality % = % test mortality - % control mortality/100 - % control mortality × 100 [ 34 ].

Distilled water + Tween 20 was used as a negative control and the larval immersion test for each concentration was repeated three times.

Adult immersion test

The AIT was performed as described by Godara et al. (2015) and the FAO (2004) [ 35 , 36 ]. The engorged female ticks were weighed and allocated to groups with 10 ticks and each group was immersed in 30 ml of the prepared concentrations, namely 0.25, 0.5, 1, 2, and 4% for 5 min. The control group was treated with distilled water + Tween20. After sieving, the retained ticks were placed onto a clean tissue paper towel for drying and kept separately in a Petri dish. The ticks were stored in an incubator at 28 °C and relative humidity of 80% to oviposit. Eggs laid by every tick were weighted and incubated to hatch [ 35 , 36 ]. There were three replications for each concentration and control.

The percentage Inhibition of Oviposition (IO) and Reproductive Index (RI) was calculated using the following formula:

Reproductive Index (RI) = Average weight of eggs laid / Average weight of live tick

Percentage inhibition of oviposition (IO) = RI of control ticks – RI of treated ticks / RI of control ticks × 100

Statistical analysis

The data were subjected to SPSS software ver. 25 (IBM corporation, USA). Statistical analysis of the data was performed using the variance analysis (ANOVA and General Linear Model) followed by Duncan's multiple range test with a probability level < 0.01.

The lethal concentrations for 50% (LC50) and 99% (LC99) with their respective 95% confidence limits (CL) were determined using regression analysis equation to the probit transformed data of mortality.

Authors' Contributions

AM and AMJ created the original idea. SG carried out the experiments, and AM, AMJ, SY, and MA directed the project. All authors analyzed and interpreted the data. AM and SG contributed to the writing of the manuscript.


The authors would like to thank the Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran for providing material and for financial support and providing the facilities that make this project possible.

Competing Interests

The authors declare that there is no conflict of interest.


  1. Nandi A, Singh H, Singh NK. Esterase and glutathione S-transferase levels associated with synthetic pyrethroid resistance in Hyalomma anatolicum and Rhipicephalus microplus ticks from Punjab, India. Exp Appl Acarol. 2015; 66(1):141-57.
  2. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary Medicine E-Book: A textbook of the diseases of cattle, horses, sheep, pigs and goats: Elsevier Health Sciences; 2006.
  3. Thomas DB, Klafke G, Busch JD, Olafson PU, Miller RA, Mosqueda J, et al. Tracking the increase of acaricide resistance in an invasive population of Cattle Fever Ticks (Acari: Ixodidae) and implementation of real-time PCR assays to rapidly genotype resistance mutations. Ann Entomol Soc Am. 2020; 113(4):298-309.
  4. Gupta S, Gupta S, Kumar S. Cypermethrin resistance in Hyalomma anatolicum and Rhipicephalus microplus ticks of arid and semi-arid zone of Haryana, a northern state of India. Int J Trop Insect Sci. 2021; 41(1):703-9.
  5. Webster A, Souza UA, Martins JR, Klafke G, Reck J, Schrank A. Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of Metarhizium anisopliae on Rhipicephalus microplus tick larvae. Exp Appl Acarol. 2018; 74(4):455-61.
  6. Sharma D, Paul Y. Preliminary and pharmacological profile of Melia azedarach L. : An overview. J App Pharm Sci 2013; 3(12):133-8.
  7. Venson I, Guzmán JS, Talavera FF, Richter H. Biological, physical and mechanical wood properties of Paraiso (Melia azedarach) from a roadside planting at Huaxtla, Jalisco, Mexico. J Trop For Sci. 2008; 20(1):38-47.
  8. Borges L, Ferri P, Silva W, Silva W, Silva J. In vitro efficacy of extracts of Melia azedarach against the tick Boophilus microplus. Med Vet Entomol. 2003; 17(2):228-31.
  9. Carpinella MC, Miranda M, Almirón WR, Ferrayoli CG, Almeida FL, Palacios SM. In vitro pediculicidal and ovicidal activity of an extract and oil from fruits of Melia azedarach L. J Am Acad Dermatol. 2007; 56(2):250-6.
  10. Sariosseiri A, Moshaverinia A, Khodaparast MHH, Kalidari GA. In vitro acaricidal effect of Melia azedarach ripe fruit extract against Dermanyssus gallinae (Acari: Dermanyssidae). Persian J Acarol. 2018; 7(2):203-208.
  11. Selvaraj M, Mosses M. Efficacy of Melia azedarach on the larvae of three mosquito species Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae). Eur Mosq Bull. 2011; 29(1):116-21.
  12. Ambrosino P, Fresa R, Fogliano V, Monti SM, Ritieni A. Extraction of azadirachtin A from neem seed kernels by supercritical fluid and its evaluation by HPLC and LC/MS. J Agric Food Chem. 1999; 47(12):5252-6.
  13. Carpinella MC, Defago MT, Valladares G, Palacios SM. Antifeedant and insecticide properties of a limonoid from Melia azedarach (Meliaceae) with potential use for pest management. J Agric Food Chem. 2003; 51(2):369-74.
  14. Nisbet AJ. Azadirachtin from the neem tree Azadirachta indica: its action against insects. An Soc Entomol Brasil. 2000; 29(4):615-32.
  15. Prakash G, Emmannuel C, Srivastava AK. Variability of azadirachtin in Azarirachta indica (neem) and batch kinetics studies of cell suspension culture. Biotechnol Bioprocess Eng. 2005; 10(3):198-204.
  16. Prakash G, Bhojwani SS, Srivastava AK. Production of azadirachtin from plant tissue culture: state of the art and future prospects. Biotechnol Bioprocess Eng. 2002; 7(4):185-93.
  17. Fernandes SR, Barreiros L, Oliveira RF, Cruz A, Prudêncio C, Oliveira AI, et al. Chemistry, bioactivities, extraction and analysis of azadirachtin: State-of-the-art. Fitoterapia. 2019; 134(1):141-50.
  18. Hosseini-Chegeni A, Hosseini R, Tavakoli M, Telmadarraiy Z, Abdigoudarzi M. The Iranian Hyalomma (Acari: Ixodidae) with a key to the identification of male species. Persian J Acarol. 2013; 2(3):503-529.
  19. Nabian S, Rahbari S, Changizi A, Shayan P. The distribution of Hyalomma spp. ticks from domestic ruminants in Iran. Med Vet Entomol 2009; 23(3):281-3.
  20. Rahbari S, Nabian S, Shayan P. Primary report on distribution of tick fauna in Iran. Parasitol Res. 2007; 101(2):175-7.
  21. Walker AR. Ticks of domestic animals in Africa: a guide to identification of species: Bioscience Reports Edinburgh; 2003.
  22. Razmi GR, Ebrahimzadeh E, Aslani M. A study about tick vectors of bovine theileriosis in an endemic region of Iran. J Vet Med. 2003; 50(6):309-10.
  23. Kadıoğlu I. Acaricidal effects of different plant parts extracts on two-spotted spider mite (Tetranychus urticae Koch). Afr J Biotechnol. 2011; 10(55):11745-50.
  24. Castillo LE, Jiménez J, Delgado M. Secondary metabolites of the Annonaceae, Solanaceae and Meliaceae families used as biological control of insects. Trop Subtrop Agroecosystems. 2010; 12(3):445-62.
  25. Isman MB. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu Rev Entomol. 2006; 51(1):45-66.
  26. Maccioni A, Falconieri D, Porcedda S, Piras A, Gonçalves MJ, Alves-Silva JM, et al. Antifungal activity and chemical composition of the essential oil from the aerial parts of two new Teucrium capitatum L. chemotypes from Sardinia Island, Italy. Nat Prod Res 2021; 35(24): 6007-6013.
  27. Yakkundi SR, Thejavathi R, Ravindranath B. Variation of azadirachtin content during growth and storage of neem (Azadirachta indica) seeds. J Agric Food Chem. 1995; 43(9):2517-19.
  28. Sousa LADD, Rocha TL, Sabóia-Morais SMT, Borges LMF. Ovary histology and quantification of hemolymph proteins of Rhipicephalus (Boophilus) microplus treated with Melia azedarach. Rev Bras Parasitol Vet. 2013; 22(3):339-45.
  29. Abdel-Ghany HS, Fahmy MM, Abuowarda MM, Abdel-Shafy S, El-Khateeb RM, Hoballah EM. In vitro acaricidal effect of Melia azedarach and Artemisia herba-alba extracts on Hyalomma dromedarii (Acari: Ixodidae): embryonated eggs and engorged nymphs. J Parasit Dis. 2019; 43(4):696-710.
  30. Borges LMF, Ferri PH, Silva WC, Silva WJ, Melo LS, Souza LAD, et al. Ação do extrato hexânico de frutos maduros de Melia azedarach (Meliaceae) sobre Boophilus microplus (Acari: Ixodidae) em bezerros infestados artificialmente. Rev Biol Trop. 2005; 34(1):53-59.
  31. Sousa LADD, Júnior HBP, Soares SF, Ferri PH, Ribas P, Lima EM, et al. Potential synergistic effect of Melia azedarach fruit extract and Beauveria bassiana in the control of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in cattle infestations. Vet Parasitol. 2011; 175(3-4):320-4.
  32. Abdel-Shafy S, Zayed A. In vitro acaricidal effect of plant extract of neem seed oil (Azadirachta indica) on egg, immature, and adult stages of Hyalomma anatolicum excavatum (Ixodoidea: Ixodidae). Vet parasitol. 2002; 106(1):89-96.
  33. Singh N, Saini S, Singh H, Sharma S, Rath S. In vitro assessment of the acaricidal activity of Piper longum, Piper nigrum, and Zingiber officinale extracts against Hyalomma anatolicum ticks. Exp Appl Acarol. 2017; 71(3):303-17.
  34. Sabatini G, Kemp D, Hughes S, Nari A, Hansen J. Tests to determine LC50 and discriminating doses for macrocyclic lactones against the cattle tick, Boophilus microplus. Vet Parasitol. 2001; 95(1):53-62.
  35. Godara R, Parveen S, Katoch R, Yadav A, Katoch M, Khajuria J, et al. Acaricidal activity of ethanolic extract of Artemisia absinthium against Hyalomma anatolicum ticks. Exp Appl Acarol. 2015; 65(1):141-8.
  36. FAO. Module 1. Ticks: Acaricide resistance; diagnosis management and prevention.